1. Field of the Invention
This invention relates generally to the field of measuring therapeutic drugs in biological samples. More specifically, it relates to a method for the enzymatic measurement of mycophenolic acid and other IMPDH inhibitors in a biological sample.
2. Description of the Related Art
The measurement of mycophenolic acid is of clinical significance. Mycophenolic acid is an immunosuppressive drug used to prevent rejection of transplanted organs, and the monitoring of mycophenolic acid is suggested to improve therapeutic efficacy and to minimize adverse side effects of the drug.
Mycophenolic acid is produced by the fermentation of several penicillium species. It has a broad spectrum of activities, specific mode of action, and is tolerable in large doses with minimal side effects, Epinette et al, Journal of the American Academy of Dermatology 17(6):962-971 (1987). Mycophenolic acid has been shown to have antitumor, antiviral, antipsoriatric, immunosuppressive and anti-inflammatory activities, Lee et al., Pharmaceutical Research 7(2):161-166 (1990), along with antibacterial and antifungal activities, Nelson, P. H. et al., Journal of medicinal Chemistry 33(2):833-838 (1990). MPA acts by inhibiting inosine-5'-monophosphate dehydrogenase (IMPDH), a key enzyme in the de novo synthesis of purine nucleotides. Since T and B lymphocytes depend largely upon this de novo synthesis, mycophenolic acid is able to inhibit lymphocyte proliferation, which is a major factor of the immune response.
Inosine-5'-monophosphate dehydrogenase (EC 1.1.1.205) catalyzes the NAD-dependent oxidation of inosine-5'-monophosphate (IMP) to xanthosine-5'-monophosphate (XMP), Magasanik, B. et al., J Biol. Chem.226:339-350 (1957) and Jackson et al., Nature 256:331-333 (1975). The enzyme follows an ordered Bi--Bi reaction sequence of substrate and cofactor binding and product release. First, IMP binds to IMPDH. This is followed by the binding of the cofactor NAD. The reduced cofactor, NADH, is then released from the product, followed by the product, XMP. This mechanism differs from that of most other known NAD-dependent dehydrogenases, which have either a random order of substrate addition or require NAD to bind before the substrate.
Two isoforms of human IMPDH, designated type I and type II, have been identified and sequenced, Collart et al., J Biol. Chem.263:15769-15772 (1988) and Natsumeda et al., J Biol. Chem.265:5292-5295 (1990). Each isoform is 514 amino acids, and both isoforms share 84% sequence identity. Both IMPDH type I and type II form active tetramers in solution, with subunit molecular weights of 56 kDa, Yamada et al., Biochemistry 27:2737-2745 (1988).
The morpholinoethyl ester of mycophenolic acid, morpholinoethyl E-6-(1,3-dihydro-4-hydroxy-6-methoxy-7-methyl-3-oxo-5-isobenzofuranyl)-4-m ethyl-4-hexenoate (MPA-M) is hydrolyzed in vivo to mycophenolic acid. Administration of mycophenolic acid in the form of this ester, also known as mycophenolate mofetil (MMF), greatly improves mycophenolic acid's bioavailability. MPA-M has a number of other favorable pharmaceutical characteristics, including its stability at pH 2-5 and its good solubility at low pH indicating rapid dissolution in the upper gastro-intestinal tract, Lee, et al., supra.
When used in combination therapy with cyclosporin A, MPA-M and cyclosporin A may have a synergistic mode of action. Cyclosporin A has a selective effect on T cells, but does not suppress B cell antibody production activity, while mycophenolic acid has an anti-proliferative effect on both T and B cells. Combined cyclosporin A/MPA-M therapy may increase survival time and allow for use of lower doses of cyclosporin A, which would reduce the side effects associated with cyclosporin A, primarily nephrotoxicity.
Mycophenolic acid is metabolized by conjugation with glucuronic acid forming mycophenolic acid glucuronide (MPAG). Two additional metabolites of MPA have been described by Schutz, E. et al., Clinical Chemistry 45(3):419-422 (1999), the 7-O-glucoside of MPA (M-1) and the acyl glucuronide of MPA (M-2). Schutz et al. measured the inhibition of recombinant human IMPDH-II by MPA, MPAG, M-1 and M-2 using purified preparations of the metabolites and found that M-2 exhibited a concentration-dependent inhibition of IMPDH-II similar to that obtained with MPA. However, different preparations of M-2 were not consistent in their ability to inhibit IMPDH-II. The metabolite M-2 is likely to be a mixture of isomers since it is known that acyl glucuronides undergo intramolecular rearrangement at physiological pH. Also, acyl glucuronides can hydrolyze back to MPA. Note that a mixture of isomers of M-2 will all equally cross react with an antibody but may not equally inhibit IMPDH. In light of the Schutz findings, it may not be surprising that measurements of MPA using the method of the present invention correlate well with results obtained via HPLC (r=0.994).
Because mycophenolic acid is a potent biologically active material, an effective assay would be useful in monitoring its bioavailability. In addition, it may be important to monitor therapeutic drug levels, i.e., optimal drug levels necessary for adequate immunosuppression. Since MPA-M is hydrolyzed to mycophenolic acid, an assay for mycophenolic acid would allow monitoring of MPA-M dosages.
The use of high-performance liquid chromatography (HPLC) to determine the concentration of mycophenolic acid in human plasma is described in Jones, C. E. et al., Journal of Chromatography B 708:229-234 (1998).
Jones et al., J Chem. Soc. (C) 1725-1737 (1970) discloses numerous transformations that mycophenolic acid undergoes when incubated with select microorganisms.
Nelson, P. H. et al., U.S. Pat. No. 4,753,935 (1988), describes the morpholinoethyl ester of mycophenolic acid, its pharmaceutical uses and post-dosage monitoring by HPLC of the recipient's plasma concentration of mycophenolic acid.
An immunoassay for mycophenolic acid using monoclonal antibodies to mycophenolic acid is described in Alexander, S. et al., PCT Publication No. WO 96/02004 (1996).
Problems of specificity with the immunoassay are discussed in Tett, S. E. et al., Clinical Chemistry 44(6):A96-A97 (1998). The authors compared results obtained on transplant patients using a commercial EMIT.RTM. immunoassay (Behring Diagnostics) with those obtained HPLC-UV and found up to 95% overestimation with the immunoassay among mid-range concentrations.
The inhibition of IMPDH by mycophenolic acid is described by Anderson, J. H. et al., Journal of Biological Chemistry 243(18):4762-4768 (1968). Inhibitors of IMPDH are also described in U.S. Pat. Nos. 5,380,879, 5,444,072 and 5,807,876 and in PCT publications WO 94/01105 and WO 94/12184.
Yatscoff, R. W. et al., Clinical Chemistry 44(2):428-432 (1998) reports on the pharmacodynamic monitoring of mycophenolic acid in human plasma by measuring the residual level of IMPDH activity present in the patient's own lymphocytes. This approach measures the patient's own biological response to the drug. It does not provide a method to directly quantitate the drug in the patient's plasma.
The cloning and expression of human IMPDH in E. coli has been described by Konno, Y. et al., J. Biol. Chem 266(1):506-509 (1991). Collart, F. R. et al., U.S. Pat. No. 5,665,583 (1997) also describe the cloning and expression in E. coli of human IMPDH.